Calcineurin inhibitory compounds and anchoring protein

ABSTRACT

The present invention provides compositions and methods useful for isolating calcineurin as well as inhibiting calcineurin activity. The compositions are peptides that contain regions that are homologous to calcineurin-binding regions of AKAP 79. Also provided are methods for determining if a cell contains a calcineurin-binding and PKA-binding anchoring protein that are useful for identifying additional proteins that bind both calcineurin and PKA.

This is a divisional application of U.S. patent application Ser. No.08/344,227, filed Nov. 23, 1994, which is pending.

FIELD OF THE INVENTION

The present invention relates generally to regulation of the phosphataseenzymatic activity of calcineurin. More particularly, the presentinvention relates to inhibition of calcineurin's phosphatase activity bycertain peptides.

BACKGROUND OF THE INVENTION

Calcineurin is a Ca² + /calmodulin-dependent protein phosphatase and isan element of many intracellular signaling pathways. Guerini and Klee,Proc. Natl. Acad. Sci. USA 86:9183-9187 (1989). The protein has beenidentified in eukaryotic cells ranging from yeast to mammals. Cyert andThorner, J. Cell. Biol., 107:841a (1989) and Klee et al., Adv. Enzymol.,61:149-200 (1984). Because calcineurin may affect many signalingpathways in the same cell, some means of specific targeting ofcalcineurin's activity is required. One cellular means for specificallytargeting enzyme activity in a cell is by compartmentalization.Compartmentalization provides a means for segregation of signalingpathways and contributes to the specificity of cellular responses todifferent stimuli. Compartmentalization of certain enzymes occurs byinteraction of the enzymes with specific anchoring proteins. Forexample, cAMP-dependent protein kinase (PKA) is anchored at specificintracellular sites by binding to A-Kinase Anchor Proteins (AKAPs).Hirsch et al., J. Biol. Chem., 267:2131-2134 (1992). cAMP activates PKAby binding to the regulatory subunits (R) of the dormant PKA holoenzymeand causes the release of the active catalytic subunit (C). Two classesof R subunit exist; RI and RII which form the type I and type II PKAholoenzymes, respectively. The subcellular distributions of these PKAisoforms appear to be distinct. The RI isoforms (RIα and RI§) arereported to be predominantly cytoplasmic and are excluded from thenuclear compartment, whereas up to 75% of the RII isoforms (RIIα orRII§) are particulate and associated with either the plasma membrane,cytoskeletal components, secretory granules, the golgi apparatus,centrosomes or possibly nuclei.

AKAPs have been identified in a variety of organisms. At least 7proteins that bind the regulatory subunit of PKA in Aplysia californica,a marine invertebrate have been identified. Cheley et al., J. Biol.Chem., 269:2911-2920 (1994). One of these proteins is enriched in crudemembrane fractions and taxol-stabilized microtubules and may thus anchormicrotubules to the cell membrane as well as bind PICA. A mammalian AKAPhas been identified that is related to microtubules;microtubule-associated protein 2 (MAP2) attaches PKA to thecytoskeleton. Threurkauf and Vallee, J. Biol. Chem., 257:3284-3290(1982) and DeCamilli et al., J. Cell Biol., 103:189-203 (1986). ThePKA-binding site on MAP2 is a 31-residue peptide in the amino-terminalregion of the molecule. Rubino et al., Neuron, 3:631-638 (1989) and Obaret al., Neuron, 3:639-645 (1989).

Another AKAP that associates with microtubules, AKAP 150, accumulates indendrites in close association with microtubules. Glantz et al., Mol.Biol. Cell, 3:1215-1228 (1992). AKAP 150 is present in several neuronalcell types and is a member of a family of AKAPs that are the principalAKAPs from mammalian brains. Other members of this family include AKAP75 found in bovine brain and AKAP 79 found in human brain. Glantz etal., J. Biol. Chem., 268:12796-12804 (1993). AKAP 75 apparently bindscytoskeletal elements through two non-contiguous regions near theN-terminus of AKAP 75. AKAP 79 is predominantly present in postsynapticdensities (PSDs) in the human forebrain. Carr et al., J. Biol. Chem.,267:16816-16823 (1992). Inhibition of binding between AKAP 79 and PKA inhippocampal neurons has been shown to inhibitalpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainateglutamate receptors. Rosenmund et al., Nature, 368:853-856 (1994).

Other AKAPs have also been characterized. Exposure of granulosa cells tofollicle-stimulating hormone and estradiol has been demonstrated toup-regulate expression of an 80 kDa AKAP. Carr et al., J. Biol. Chem.,268:20729-20732 (1993). Another AKAP, Ht31, has been cloned from a humanthyroid cDNA library. Carr et al., J. Biol. Chem., 267:13376-13382(1992). Another AKAP, AKAP 95, changes its intracellular location duringthe cell cycle. AKAP 95 is an integral nuclear protein duringinterphase, but becomes associated with cytoplasmic PKA when the nuclearmembrane breaks down during mitosis. This suggests that AKAP 95 couldplay a role in targeting activity of certain isoforms of PKA duringcAMP-responsive events linked to the cell cycle. Coghlan et al., J.Biol. Chem., 269:7658-7665 (1994). Other known AKAPs include an 85 kDaAKAP which links PKA to the Golgi apparatus (Rios et al., EMBO J.,11:1723-1731 (1992)) and a 350 kDa AKAP that binds PKA to centromeres(Keryer et al., Exp. Cell Res., 204:230-240 (1993)).

The known AKAPs share a common PKA-binding feature. Although the primarystructure of the AKAPs is not conserved, each has a secondary structuremotif that includes an amphipathic helix region. Scott and McCartney,Mol. Endo., 8:5-11 (1994). Binding of AKAPs to the regulatory subunit ofPKA is blocked by a peptide that mimics this helical structure of thePKA binding region of AKAPs. Disruption of the peptide's helicalstructure by an amino acid substitution abolishes the PKA-AKAP bindingblock (Carr et al., J. Biol. Chem., 266:14188-14192 (1991)),demonstrating that PKA binding occurs in the amphipathic helix of AKAPsand is governed by the secondary structure of the AKAP molecules. Thisprovides a means for segregation of a kinase that, like calcineurin, iscommon to many signaling pathways yet may act in a pathway-specificmanner.

One specific action of calcineurin is participation in T cellactivation. Clipstone and Crabtree, Nature, 357:695-697 (1992) andO'Keefe et al., Nature, 357:692-694 (1992). In T cells, calcineurinparticipates in regulation of IL-2 expression following T cellstimulation. Weiss and Littman, Cell, 76:263-274 (1994). Nuclear factorof activated T cells (NFAT_(p)) has been shown to be a substrate forcalcineurin phosphatase activity. It has been suggested that, followingT cell stimulation, calcineurin-mediated NFAT_(p) dephosphorylationallows translocation of NFAT_(p) from the cytoplasm to the nucleus whereNFAT_(p) interacts with Fos and Jun to induce expression of the IL-2gene. Jain et al., Nature, 365:352-355 (1993).

Calcineurin's role in T cell activation provides a target fortherapeutic intervention into T cell-mediated disorders and medicationshave been developed that inhibit calcineurin. Two calcineurin-inhibitingdrugs, cyclosporin A (cyclosporin) and FK506, have been used in theclinic. Thomson and Starzl, Immunol. Rev., 136:71-98 (1993). Bothcyclosporin and FK506 inhibit calcineurin only after binding to distinctintracellular proteins known as immunophilins (cyclophilin and FKBP 12,respectively). Schreiber and Crabtree, Immunology Today, 13:136-142(1992). Thus, cyclosporin and FK506 act as prodrugs. Following bindingto their respective immunophilins, the drug/immunophilin complexes bindcalcineurin, thereby inhibiting the phosphatase activity.

Calcineurin inhibition has been most effectively exploited in thetreatment of graft rejection following organ transplant. Cyclosporin andFK506 have been employed following renal, hepatic, cardiac, lung, andbone marrow transplants. The Canadian Multicentre Transplant StudyGroup, N. Engl. J. Med., 314:1219-1225 (1986); Oyer et al., TransplantProc., 15:Suppl 1:2546-2552 (1983); Starzl et al., N. Engl. J. Med.,305:266-269 (1981); The Toronto Lung Transplant Group, JAMA,259:2258-2262 (1988); and Deeg et al., Blood, 65:1325-1334 (1985). Theuse of these medications has significantly prolonged graft survival andlessened morbidity following transplant. Najarian et al., Ann. Surg.,201:142-157 (1985) and Showstack et al., N. Engl. J. Med., 321:1086-1092(1989).

Cyclosporin also has been used in a variety of autoimmune-relateddiseases. Uveitis generally improves within a few weeks of therapy, butquickly relapses after cyclosporin is discontinued. Nussenblatt et al.,Am J. Ophthalmol., 96:275-282 (1983). Similarly, psoriasis generallyimproves with cyclosporin therapy, but quickly relapses after treatment.Ellis et al., JAMA, 256:3110-3116 (1986). "Honeymoon" periods of insulinindependence may be induced and prolonged in both new onset Type I andType II diabetes mellitus when cyclosporin is administered within twomonths of insulin therapy. Feutren et al., Lancet, 2:119-124 (1986) andBougneres et al., N. Engl. J. Med., 318:663-670 (1988). A variety ofnephropathies, including minimal-change focal and segmental, membranous,and IgA-mediated nephropathies, may also be sensitive to cyclosporin,although observed reductions in proteinuria may be due to a decrease inthe glomerular filtration rate and not healing of the basement membrane.Tejani et al., Kidney Intl., 29:206 (1986). Cyclosporin administrationalso has a dose-dependent effect on rheumatoid arthritis, although suchtreatment is associated with a high incidence of nephrotoxicity. F.oslashed.rre et al., Arthritis Rheum., 30:88-92 (1987).

As mentioned above, cyclosporin has been associated with nephrotoxicity.Mason, Pharmacol. Rev., 42:423-434 (1989). Depressed renal functionoccurs in virtually all patients treated with cyclosporin. Kahan, N.Engl. J. Med., 321:1725-1738 (1989). This can generally be reversed bycessation of cyclosporin therapy. Unfortunately, in organ graftrecipients substitution of other commonly used immunosuppressives forcyclosporin carries a high risk of graft rejection. In renal transplantpatients this can require reinstitution of dialysis. In patients thathave received hearts, lungs, or livers, graft rejection can be fatal.Although less common than nephrotoxicity, neurotoxicity andhepatotoxicity are also associated with cyclosporin therapy. de Groen etal., N. Engl. J. Med., 317:861-866 (1987) and Kahan et al.,Transplantation, 43:197-204 (1987).

Although there is less clinical experience, significant toxicity hasalso become apparent in the use of FK506. Like cyclosporin, FK506 isassociated with significant nephrotoxicity. Peters et al., Drugs,4:746-794 (1993). The clinical presentation, lesion morphology, andincidence are approximately equivalent to those of cyclosporin.McCauley, Curr. Op. Nephrol. Hyperten., 2:662-669 (1993). Neurotoxicityhas also been associated with FK506. Eidelman et al., Transplant. Proc.,23:3175-3178 (1991) and Fung et al., Transplant. Proc., 23:3105-3108(1991). In contrast to cyclosporin, FK506 has a hepatotrophic, ratherthan hepatotoxic, effect. Peters et al., supra.

In view of the significant potential toxicity of immunosuppressiveagents, such as cyclosporin and FK506, it is clear that there is a needin the art for additional agents that inhibit calcineurin. These agentswould preferably be associated with fewer toxic side effects thanpresently available agents and thus could provide an advance inimmunosuppressive therapy.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatcalcineurin binds AKAP 79. By binding both PKA and calcineurin, AKAP 79co-localizes a kinase and a phosphatase that may regulate flux through aspecific signaling pathway. The present invention accordingly providescompositions and methods for isolating calcineurin as well as forinhibiting calcineurin activity in a cell. The isolation methodscomprise contacting a cellular fraction with AKAP 79 or acalcineurin-binding fragment thereof which has been immobilized to asolid substrateand then elutin calcineurin therefrom. The calcineurininhibiting methods comprise contacting the cell with AKAP 79 or acalcineurin-binding fragment peptide thereof. Preferrably, thecalcineurin-binding peptide does not also bind PKA. Preferred peptidescomprise the following amino acid sequence:

    Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro        (SEQ ID NO:1).

Alternative peptides useful in the practice of the calcineurininhibiting methods of the present invention include:

    Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro-Leu-Gln (SEQ ID NO:2)

    and

    Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro-Phe-Lys (SEQ ID NO:3).

These peptides are homologous to amino acid sequences of AKAP 79 thatbind calcineurin. Although the peptides are similar to the calcineurinbinding region of FKBP12, unlike calcineurin inhibition by theFK506/FKBP12 complex, the peptides inhibit calcineurin activity withoutrequiring interaction with another molecule.

The peptides may be modified to facilitate passage into the cell, suchas by conjugation to a lipid soluble moiety. For example, the peptidesmay be conjugated to myristic acid. Alternatively, the peptides may bepackaged in liposomes that may fuse with cell membranes and release thepeptides into the cells.

Another aspect of the present invention are methods for determining if acell contains a calcineurin-binding and PKA-binding anchoring protein.The methods generally comprise lysing the cell to form a lysate;incubating the lysate with a solid support, which solid support hascalcineurin molecules immobilized thereon; washing the lysate from thesolid support; contacting the solid support with a labeled PKAregulatory subunit, washing unbound regulatory subunit from the solidsupport; detecting label remaining on the solid support; and determiningtherefrom the presence of a calcineurin-binding and PKA-bindinganchoring protein in the cell. Alternatively, the PKA regulatory subunitmay immobilized on the solid support and calcineurin may be the labeledmolecule. Generally, the PKA regulatory subunit will be an RII subunit.

These methods are useful for identifying additional proteins that bindboth PKA and calcineurin. Identification of other such proteins mayprovide tissue specific targets for therapeutic intervention.

Also comprehended by the present invention are methods for identifyingcompounds that modulate binding between calcineurin and a calcineurinanchoring protein. Either calcineurin or the anchoring protein may bebound to a solid substrate. The unbound binding partner is detectablylabeled. The binding partners are incubated in the presence of a testcompound. The effect of the test compound on binding between calcineurinand the calcineurin anchoring protein is determined by observing theamount of label bound to the immoblized binding partner. A reduction inthe amount of label bound in the presence of the test compound comparedto the amount of label bound in the absence of the test compoundindicates that the test compound is an inhibitor of binding betweencalcineurin and the calcineurin anchoring protein. Other assays, such asscintillation proximity assays may also be employed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(a-c) illustrate inhibition of calcineurin phosphatase activityby full-length AKAP 79 and a calcineurin-binding fragment of AKAP 79.

FIGS. 2(a-c) illustrate subcellular localization of type II PKA andcalcineurin as well as the co-localization of type II PKA andcalcineurin.

FIG. 3 illustrates homology between clone 11.1 and human calcineurinisoform 11.1.

DETAILED DESCRIPTION OF THE INVENTION

The peptides employed in the methods of the present invention may besynthesized in solution or on a solid support in accordance withconventional techniques as described in Stewart and Young, Solid PhasePeptide Synthesis, 2nd ed., Pierce Chemical Company, (1984) or Tam etal., J. Am. Chem. Soc., 105:6442 (1983), both of which are incorporatedherein by reference. The peptides may be myristoylated by standardtechniques as described in Eichholtz et al., J. Biol. Chem.,268:1982-1986 (1993), incorporated herein by reference. Encapsulation ofthe peptides in liposomes may also be performed by standard techniquesas generally described in U.S. Pat. Nos. 4,766,046; 5,169,637;5,180,713; 5,185,154; 5,204,112; and 5,252,263 and PCT PatentApplication No. 92/02244, each of which is incorporated herein byreference.

The following examples are offered by way of illustration and not oflimitation.

EXAMPLE 1

This example demonstrates the naturally-occurring association ofcalcineurin with AKAP 79 and PKA. AKAP 79 thus functions to co-localizeboth a ubiquitous kinase and ubiquitous phosphatase. Thisco-localization may provide for specific regulation of enzymes insignaling pathways through phosphorylation or dephosphorylation of theenzymes.

Immunoprecipitation of calcineurin (CaN) from a calmodulin-agarosepurified bovine brain extract was achieved using affinity-purifiedantibodies specific for either CaN A or CaN B as generally described inHarlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1988), except a final wash using bufferA (10 mM HEPES pH 7.9, 1.5 mM MgCl, 10 mM KCl, 1 mM PMSF and 10 μMIBMX)+0.4M NaCl was included. PKA activity was measured as described inScott et al., Proc. Natl. Acad. Sci. USA, 82:4379-4383 (1985),incorporated herein by reference, after elution of the immunoprecipitatewith 0.1 mM cAMP. Phosphorylation of immunoprecipitated proteins wasinitiated by addition of 0.1 mM ³² P-ATP (1.5×10⁵ cpm/nmol) and, after30 min at 30° C., reactions were terminated by addition of SDS-loadingbuffer and subjected to SDS-PAGE. PKA R-subunit was purified from the30-60% (NH₄)₂ SO₄ fraction of brain extract using cAMP-agarose by themethods described in Coghlan et al., J. Biol. Chem., 269:7658-7665(1994) (incorporated herein by reference), except protein was elutedwith 0.5 mM Ht31 peptide (SEQ ID NO:4). Western blots and PKA RIIoverlays were performed as described in Coghlan et al., supra.

Kinase activity was detected in the calmodulin purified extract, wasenriched 123±3.6 fold (±standard deviation; n=3) in the CaNimmunoprecipitate, and was specifically inhibited by a peptide thatinhibits PKA kinase activity, PKI peptide (SEQ ID NO:5), indicating thatthe catalytic (C) subunit of PKA was a component of the isolatedcomplex. The bovine homologue of AKAP 79 (AKAP 75) and RII, bothsubstrates for the C subunit, were also present in the immunoprecipitateand were phosphorylated upon addition of cAMP and ³² P-ATP. Incomplementary experiments, R subunits of PICA were isolated from crudeextracts of bovine brain by affinity chromatography on cAMP-agarose.Treatment of the affinity column with Ht31 peptide specifically elutedAKAP 75 from the cAMP-bound RII and also released both CaN A and Bsubunits. Approximately 5% of the total CaN present in the lysate wasfound to be associated with AKAP 75 and RII as detected on westernblots. Combined, these results suggest simultaneous association of PKAand CaN with the AKAP.

EXAMPLE 2

This example demonstrates inhibition of calcineurin's phosphataseactivity by peptides from AKAP 79.

To determine whether AKAP 79 peptide binding was inhibitory, calcineurin(CaN) activity was assayed in the presence of recombinant AKAP 79.Briefly, recombinant AKAP 79 was expressed in E. coli as described inCarr et al., J. Biol. Chem., 267:16816-16823 (1992), incorporated hereinby reference. CaN and the constitutively active truncation mutant CaN₄₂₀(a truncated, Ca²⁺ /calmodulin independent constitutively active form ofCaN (Perrino et al., J. Biol. Chem., in press)) were expressed in Sf9cells and purified on calmodulin-Sepharose as described in Perrino etal., J. Biol. Chem., 267:15965-15969 (1992), incorporated herein byreference. Phosphatase activity toward ³² P RII peptide substrate wasmeasured as described in Perrino et al., supra. CaN (30 nM), calmodulin(100 nM) and ³² P RII peptide (22 μM) were incubated with AKAP 79protein and AKAP 79 peptide (SEQ ID NO:1-amino acids 81-102) over theindicated range of concentrations indicated in FIG. 1B. Calmodulin wasomitted from CaN₄₂₀ assays. ³² P released from the substrate wasmeasured in triplicate samples in three separate experiments byscintillation counting. The inhibition constant (K_(i)) of recombinantAKAP 79 for CaN was determined by linear regression analysis of data.K_(i) values for AKAP 79 peptide were estimated by determining the IC₅₀using a fixed substrate concentration at K_(m) (42 μM).

FIG. 1A illustrates a Lineweaver-Burk plot of AKAP 79 inhibition of bothfull-length CaN (Ca²⁺ /calmodulin dependent) (circles) and CaN₄₂₀(squares) in a non-competitive manner with respect to phosphorylated RIIpeptide substrate. The open symbols represent phosphatase activity inthe absence of AKAP 79 and the fried symbols represent phosphataseactivity in the presence of AKAP 79. The synthetic peptide correspondingto the AKAP 79 peptide inhibited both full-length CaN (filled circles)and CaN₄₂₀, whereas the Ht31 peptide was not an inhibitor of CaN (FIG.1B). The observed inhibition was specific for calcineurin; the AKAP 79peptide did not significantly affect the activity of proteinphosphatases 1 (open diamonds) or 2A (crosses) at peptide concentrationsas high as 0.4 mM. Although CaN-binding sites on AKAP 79 and FKBP-12 aresimilar, their differences may have functional significance: FK506 (2μM) did not affect the potency of inhibition and recombinant AKAP 79 didnot display peptidyl prolyl isomerase activity toward a fluorescentpeptide substrate. Further, the CaN B subunit which is required forFK506/FKBP interaction with the CaN A subunit is not required forinteraction of AKAP 79 with the CaN A subunit. Also, while theFK506/FKBP interaction with CaN A is calcium/calmodulin dependent, theAKAP 79 inhibition of calcineurin activity is calcium/calmodulinindependent. Collectively, these findings suggest that CaN in itsinactive state is localized by AKAP 79 in a manner analogous toAKAP-bound PKA.

EXAMPLE 3

This example demonstrates subcellular distribution of type II PKA andcalcineurin in tissue.

The subcellular location of many protein kinases and proteinphosphatases is defined by association with targeting subunits. AKAP 79represents a novel member of this class of regulatory proteins as itserves a bifunctional role in localizing both PKA and CaN.

Cells were cultured, formalin-fixed, and immunostained as described inRosenmund et al., Nature, 368:853-856 (1994). FITC-conjugated anti-goatsecondary antisera was used for RII staining. Biotinylated anti-rabbitsecondary antisera and streptavidin-Texas-Red (Jackson) were used instaining for CaN. Images were obtained using a Biorad MRC-600 confocallaser scanning system (A1 and A2 filters) with a Nikon optiphot 2microscope equipped with 60× planappo chromat (1.6 NA) oil immersionlens. Confocal sections were between 1.5 and 2 μm absolute thickness.

AKAP 79 homologues were observed in bovine, porcine, rabbit, and murinebrain. This indicates that co-localization of PKA and CaN may be auniversal phenomenon that adapts neurons for specific signaltransduction events. Using immunocytochemical methods, the subcellulardistribution of type II PKA and CaN was examined in cultured hippocampalneurons. The staining patterns for RII (green label in FIG. 2A) and CaN(red label in FIG. 2B) were regionally dispersed and overlapped in theneurites (RII is red and CaN is green in FIG. 2C). These findings areconsistent with co-localization of type II PKA and CaN by the AKAP andsuggest a role for the ternary complex in regulating synaptictransmission. This is consistent with experiments demonstratingco-localization of RII and AKAP 79 in these cells, and by studiesshowing that AKAP 79, type II PKA and CaN are components of postsynapticdensities. Potential substrates for the localized ternary transductioncomplex may include AMPA/kainate receptors, which are modulated byAKAP-targeted PKA.

EXAMPLE 4

This example demonstrates interaction between AKAP 79 and calcineurin ina yeast dihybrid assay. Employing AKAP 79 as the "bait", calcineurinencoded by cDNA from a murine T cell library was found to bind toAKAP79.

The assay was performed as generally described in Durfee, et al., Genesand Development 7:555-567 (1993), incorporated herein by reference. The"target" and "bait" were two plasmids, each containing part of the Gal-4transcription factor. The "bait" plasmid (pAS1) was a 2 micron basedplasmid with an ADH promoter linked to the Gal-4 DNA binding subunit [bp1-147 as described in Keegan et al., Science, 231:699-704 (1986),incorporated herein by reference], followed by a hemagglutin (HA) tag,polyclonal site and an ADH terminator. Selection was maintained usingSC-Trp media. The "target" construct was a leu2, 2 micron based plasmidcontaining an ADH promoter and terminator with the Gal-4 transcriptionactivation domain II [amino acids 768-881 as described in Ma andPtashne, Cell, 48:847-853 (1987), incorporated herein by reference]followed by a multiple cloning site. This vector, pACT, was utilized inthe construction of a mouse T cell cDNA fusion library. Saccharomycescerevisiae y190 used in the screening was designed with two reportergenes integrated into its genome. The reporter genes are under controlof a Gal-1 promoter containing Gal-4 binding sites. If the proteinsencoded by the bait plasmid and the target plasmid associate, the Gal-4transcription factor subunits are brought together and function toinitiate transcription of the reporter genes.

A 1.3 Kb Nco1/BamH1 fragment containing the coding region of AKAP 79 wasisolated from a pET11d backbone and ligated to pAS1 to act as "bait" forthe screen. One μg of this construct was transformed into y190 MATa andy190 MATα using a standard lithium acetate-PEG transformation protocol.Four isolates of each mating type (y190A pASI AKAP 79 1-4 and y190α pAS1AKAP 79 1-4) were tested for their ability to interact with a fusionconstruct pACT-RII which contains the regulatory subunit (RII aminoacids 1-89) of PKA. This was achieved by mating the strains on YEPD (1%Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose, and 2% Bacto agar)overnight at 30° C. and then selecting for diploids on SC-Leu-Trpplates. The E. coli lac Z gene acting as the reporter could then beassayed for β-galactosidase activity. The mated strains were replicatedto SC-Leu-Trp plates that had been overlayed with Hybond-N filters(Amersham) and grown overnight. The filters were placed in liquidnitrogen for one minute to crack open the yeast. A 3MM paper disc wassaturated with approximately 3 ml 0.1% X-gal in 60 mM Na₂ HPO₄, 40 mMNaH₂ PO₄, 10 mM KCl and 10 mM MgSO₄. The lysed yeast filter was placedon top of the disc and allowed to develop at 30° C. for approximately1-2 hours. Diploid strains containing both pAS1 AKAP 79 and pACT RIIfusions that were positive for β-gal activity were indicated by miningthe yeast patch a blue color. As a control, the bait AKAP 79 plasmidremained white when mated with an empty pACT control.

Detection of the Gal-4 AKAP 79 fusion protein was achieved by growingy190A AKAP 79 (isolates 1 and 2) and y190a AKAP 79 (isolates 1 and 2) toa density of 2×10⁷ cells/ml in 50 ml SC-Trp media. Cells were pelletedat 3000×g for 10 minutes and lysed with 200 μl glass beads (size 425-600microns) in 25 mM Tris pH8, 5 mM EDTA, 5 mM EGTA, 2 mM O-phenanthroline,1 mM DTT, 25 μM AEBSF, 1 mM Benzanidine, 1 μg/ml PLACC (Pepstalin,Leupeptin, Aprotinin, Calpain I and ID, and 20 μg/ml Bestantin lysisbuffer. Cells were alternately vortexed for one minute and iced for oneminute for a total of 24 minutes (12 cycles). Protein concentrationswere determined and 30 μg of total protein was loaded onto 10% SDS-PAGEgel. The gel was wet transferred to Immobilon-P (Millipore) and detectedby standard procedures using an anti-HA monoclonal antibody 12CA5 (BabCo., Berkeley, Calif.) and goat anti-mouse IgG alkaline phosphataseconjugated secondary antiserum (Biorad, Hercules, Calif.). A Gal-4 AKAP79 fusion protein of approximately 100 kDa was readily detectableindicating the correct size product was present within these strains.

y190A pAS1 AKAP 79 isolate 1 was chosen to screen a pACT murine T cellcDNA library. A 500 ml SC-Trp culture (OD₆₀₀ =0.6-0.8) was harvested,washed with 100 ml distilled water, and repelleted. The pellet wasbrought up in 50 ml LiSORB (100 mM lithium acetate, 10 mM Tris pH8, 1 mMEDTA pH8, and 1M Sorbitol), transferred to a 1 liter flask and shaken at220 RPM for an incubation of 30 min at 30° C. The cells were thenpelleted and resuspended with 625 μl LiSORB, and held on ice whilepreparing the DNA.

The DNA was prepared for transformation by boiling 400 μl 10 mg/mlSalmon sperm DNA for 10 min after which 500 μl LiSORB was added andallowed to slowly cool to room temperature. DNA from the Mu T celllibrary was added (40-50 μg) from a 1 mg/ml stock. The iced yeastculture was dispensed into 10 Eppendorf tubes with 120 μl of preparedDNA. The tubes were incubated at 30° C. at 220 RPM. After 30 minutes,900 μl of 40% PEG₃₃₅₀ in 100 mM Li acetate, 10 mM Tris pH 8 and 1 mMEDTA pH 8 was mixed with each culture and returned to incubate for anadditional 30 min. The samples were then pooled and a small aliquot (5μl) was removed to test for transformation efficiency and plated onSC-Leu-Trp plates. The remainder of the cells were added to 100 mlSC-Leu-Trp-His media and grown for 1 hr at 30° C. with shaking at 220RPMS. Harvested cells were resuspended in 5.5 ml SC-Leu-Trp-His+50 mM3AT (3-amino triazole) media and 300 μl aliquots plated on 150 mmSC-Leu-Trp-His+50mM 3AT and left to grow for 1 week at 30° C.

After four days, titer plates were counted and 1.1×10⁵ colonies werescreened. Large scale β-gal assays were performed on library plates andten positive clones were isolated for single colonies. One of thesecolonies grew substantially larger than the rest, and was termed clone11.1. Total yeast DNA was prepared from these strains and leu2 plasmidDNA was isolated. The "rescued" plasmid was used to retransform theoriginal y190A pAS1 AKAP 79 bait strain and y190a. Only clone 11.1remained positive for β-galactosidase activity in y190A pAS1 AKAP 79.y190a containing pACT clone 11.1 remained white serving as a negativecontrol.

Restriction digestion with endonuclease Xho1 released a 2.3 Kb insertand the plasmid was sequenced in the forward and reverse directions.Reactions from the Dye Deoxy Terminator Cycle Sequencing Kit (AppliedBiosystems, Inc. Foster City, Calif.) using symmetric polymerase chainreaction (PCR) on double stranded templates were analyzed on an ABI 373Aautomated sequencer (Applied Biosystems, Inc.). Sequence from clone 11.1revealed an open reading frame 487 aa long (SEQ ID NO:6) which wascorrectly fused to the Gal-4 activation domain of pACT. The NIH sequencedatabase was searched and the sequence was found to be closelyhomologous to the human calmodulin dependent protein phosphatase,calcineurin. Computer analysis between clone 11.1 and the human isoformA1 showed an 80% identity on the nucleic acid level and 93% identity onthe amino acid level (FIG. 3). The first 10aa and an 18aa insert in thehuman sequence are not present in the mouse 11.1 sequence. Clone 11.1 isclosely related to the mouse calcineurin A b sequence, but is distinctlydissimilar at the carboxy-terminus. Likewise the human calcineurin A1and human calcineurin A2 isoforms are closely homologous but aredistinct from each other at their 3' ends.

Specificity of the AKAP 79-calcineurin interaction was demonstrated bymating the calcineurin pACT containing strain with other unrelated baitstrains. Crosses were performed as described above with strainscontaining pAS1 fused to RII (1-89), casein kinase 1, phosphodiesterase32 (HDUN2) and AKAP Ht31. β-galactosidase activity was negative in allof these diploid strains.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich the invention pertains. All publications, patents, and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated herein byreference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is understood that certain changes and modificationsare within the level of skill in the an and are within the scope of theappended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgArgLysArgSerGlnSerSerLysGluGluLysPro                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgArgLysArgSerGlnSerSerLysGluGluLysProLeuGln                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgArgLysArgSerGlnSerSerLysGluGluLysProPheLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspLeuIleGluGluAlaAlaValSerArgIleValAspAlaValIle                              151015                                                                        GluGluValLysAlaAlaGlyAla                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlyArgArgAsnAlaIleHisAspIle                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2257 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1461                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCGCCCCCGCCCCCGCCCCCACCGCCCCCTCTCGGGGCCGACCGCGTC48                            ProProProProProProProProProProLeuGlyAlaAspArgVal                              151015                                                                        GTCAAAGCTGTTCCTTTTCCCCCAACTCATCGGCTGACATCTGAAGAA96                            ValLysAlaValProPheProProThrHisArgLeuThrSerGluGlu                              202530                                                                        GTGTTTGATATGGATGGGATACCCAGGGTTGATGTTCTGAAGAACCAC144                           ValPheAspMetAspGlyIleProArgValAspValLeuLysAsnHis                              354045                                                                        TTGGTAAAAGAAGGGCGGGTGGATGAAGAAATTGCACTAAGAATTATC192                           LeuValLysGluGlyArgValAspGluGluIleAlaLeuArgIleIle                              505560                                                                        AATGAGGGTGCTGCCATACTTCGGCGGGAGAAAACCATGATAGAAGTA240                           AsnGluGlyAlaAlaIleLeuArgArgGluLysThrMetIleGluVal                              65707580                                                                      GAAGCTCCAATTACAGTGTGTGGTGACATCCATGGCCAATTTTTTGAT288                           GluAlaProIleThrValCysGlyAspIleHisGlyGlnPhePheAsp                              859095                                                                        CTGATGAAACTTTTTGAAGTAGGAGGATCACCTGCTAATACACGATAC336                           LeuMetLysLeuPheGluValGlyGlySerProAlaAsnThrArgTyr                              100105110                                                                     CTTTTTCTTGGTGATTATGTGGACAGAGGTTATTTTAGTATAGAGTGT384                           LeuPheLeuGlyAspTyrValAspArgGlyTyrPheSerIleGluCys                              115120125                                                                     GTCTTATATTTATGGGTCTTGAAGATTCTATACCCAAGCACATTATTC432                           ValLeuTyrLeuTrpValLeuLysIleLeuTyrProSerThrLeuPhe                              130135140                                                                     CTTCTGAGAGGCAACCATGAATGCAGACACCTTACTGAATATTTTACC480                           LeuLeuArgGlyAsnHisGluCysArgHisLeuThrGluTyrPheThr                              145150155160                                                                  TTTAAGCAGGAATGTAAAATTAAATATTCAGAAAGAGTCTATGAAGCT528                           PheLysGlnGluCysLysIleLysTyrSerGluArgValTyrGluAla                              165170175                                                                     TGTATGGAGGCTTTTGACAGCTTGCCCCTTGCTGCACTTCTAAACCAA576                           CysMetGluAlaPheAspSerLeuProLeuAlaAlaLeuLeuAsnGln                              180185190                                                                     CAATTTCTTTGTGTTCATGGTGGACTTTCACCAGAAATACACACACTG624                           GlnPheLeuCysValHisGlyGlyLeuSerProGluIleHisThrLeu                              195200205                                                                     GATGATATTAGGAGATTAGATAGATTTAAAGAGCCACCTGCATTTGGA672                           AspAspIleArgArgLeuAspArgPheLysGluProProAlaPheGly                              210215220                                                                     CCAATGTGTGACTTGCTATGGTCTGATCCTTCTGAAGACTTTGGAAAT720                           ProMetCysAspLeuLeuTrpSerAspProSerGluAspPheGlyAsn                              225230235240                                                                  GAAAAATCACAAGAACATTTTAGTCATAATACAGTTCGAGGATGTTCT768                           GluLysSerGlnGluHisPheSerHisAsnThrValArgGlyCysSer                              245250255                                                                     TATTTTTATAACTATCCAGCAGTGTGTGAATTTTTGCAAAACAATAAT816                           TyrPheTyrAsnTyrProAlaValCysGluPheLeuGlnAsnAsnAsn                              260265270                                                                     TTGTTATCGATTATTAGAGCTCATGAAGCTCAAGATGCAGGCTATAGA864                           LeuLeuSerIleIleArgAlaHisGluAlaGlnAspAlaGlyTyrArg                              275280285                                                                     ATGTACAGAAAAAGTCAAACTACAGGGTTTCCTTCATTAATAACAATT912                           MetTyrArgLysSerGlnThrThrGlyPheProSerLeuIleThrIle                              290295300                                                                     TTTTCGGCACCTAATTACTTAGATGTCTACAATAATAAAGCTGCTGTA960                           PheSerAlaProAsnTyrLeuAspValTyrAsnAsnLysAlaAlaVal                              305310315320                                                                  CTAAAGTATGAAAATAATGTGATGAACATTCGACAGTTTAATTGCTCT1008                          LeuLysTyrGluAsnAsnValMetAsnIleArgGlnPheAsnCysSer                              325330335                                                                     CCACATCCTTATTGGTTGCCCAATTTTATGGATGTCTTTACATGGTCC1056                          ProHisProTyrTrpLeuProAsnPheMetAspValPheThrTrpSer                              340345350                                                                     TTACCATTTGTTGGAGAAAAAGTGACAGAAATGTTGGTAAATGTTCTG1104                          LeuProPheValGlyGluLysValThrGluMetLeuValAsnValLeu                              355360365                                                                     AGTATTTGTTCTGATGATGAACTAATGACAGAAGGTGAAGACCAGTTT1152                          SerIleCysSerAspAspGluLeuMetThrGluGlyGluAspGlnPhe                              370375380                                                                     GATGTAGGTTCAGCTGCAGCCCGGAAAGAAATCATAAGAAACAAGATC1200                          AspValGlySerAlaAlaAlaArgLysGluIleIleArgAsnLysIle                              385390395400                                                                  CGAGCAATTGGCAAGATGGCAAGAGTCTTCTCTGTTCTCAGGGAGGAG1248                          ArgAlaIleGlyLysMetAlaArgValPheSerValLeuArgGluGlu                              405410415                                                                     AGTGAAAGCGTGCTGACACTCAAGGGCCTGACTCCCACAGGGATGTTG1296                          SerGluSerValLeuThrLeuLysGlyLeuThrProThrGlyMetLeu                              420425430                                                                     CCTAGTGGAGTGTTGGCTGGAGGACGGCAGACCTTGCAAAGTGGTAAT1344                          ProSerGlyValLeuAlaGlyGlyArgGlnThrLeuGlnSerGlyAsn                              435440445                                                                     GATGTTATGCAACTTGCTGTGCCTCAGATGGACTGGGGCACAACTCAC1392                          AspValMetGlnLeuAlaValProGlnMetAspTrpGlyThrThrHis                              450455460                                                                     TCTTTTGCTAACAATACACATAATGCATGCAGGGAACTCCTTCTGCTT1440                          SerPheAlaAsnAsnThrHisAsnAlaCysArgGluLeuLeuLeuLeu                              465470475480                                                                  TTTAGTTCCTGTCTTAGCAGCTGACATATGCAGGGTATTATGTGATAGGCA1491                       PheSerSerCysLeuSerSer                                                         485                                                                           TCTGATTAGTACCTGGCCAGGGCATAATATTGATAGAACAAGTTGTCTTTTAACTGAAAA1551              TAACAATCAGTTTCCCAGATTTTCATAAGGTGATATGGGGAGCAGCTCATGTCATAATTC1611              CGAAATATTTATTCATTTGTTTAATGCACCCCTTTCTTTCAAAAGCCTCAGTCAAGAATG1671              TGAATCAGGGATATATCTATATATCTATTTACACACATACATAAATATATATAACTAAAA1731              TGGAAATGTAATTCCGAGTTTCTTACTTTTAAAATTTACGTAATTGTATTAGATTTTGCT1791              TATGTTTTCAAGTATTTATTTTTTGAGTTAAAATTCTGCTTAGGCCCCAAAACTTCCTTT1851              ATGCACTCATTTGCCAAAAGATTTATGCTAAATTTTGTACCCTGGTAAATGATTAGAGTT1911              TGTTTTCTGTGGTGTTTGTCAAACGTTCTATGTATAATTGACTGTCTGTAACATGCTGTT1971              TCCTTCCTCTGCAGATATAGCTGCTTTCCTAAATCTGTCTGTCTTTCTTTAGGATAGCTG2031              TATGTCTGTAAATATATGTTCAATTAAATTACTCTATCAGACGCTTGTCTGTCTTTTGAT2091              GTAGAAGCAACTTTGTAGCACCTTGATTTTAGGTTTGCTGCATTTGTTGCTGCACTTGGT2151              TCAGTCTGAATATGAATGTAACATTAGATATTGAGCTATTGTTATAAAGGGTTGAATTTA2211              AATCATGTAAGTCAAAATTGAAAGGGTGTTATAAAGTGTGCCTTTA2257                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 487 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ProProProProProProProProProProLeuGlyAlaAspArgVal                              151015                                                                        ValLysAlaValProPheProProThrHisArgLeuThrSerGluGlu                              202530                                                                        ValPheAspMetAspGlyIleProArgValAspValLeuLysAsnHis                              354045                                                                        LeuValLysGluGlyArgValAspGluGluIleAlaLeuArgIleIle                              505560                                                                        AsnGluGlyAlaAlaIleLeuArgArgGluLysThrMetIleGluVal                              65707580                                                                      GluAlaProIleThrValCysGlyAspIleHisGlyGlnPhePheAsp                              859095                                                                        LeuMetLysLeuPheGluValGlyGlySerProAlaAsnThrArgTyr                              100105110                                                                     LeuPheLeuGlyAspTyrValAspArgGlyTyrPheSerIleGluCys                              115120125                                                                     ValLeuTyrLeuTrpValLeuLysIleLeuTyrProSerThrLeuPhe                              130135140                                                                     LeuLeuArgGlyAsnHisGluCysArgHisLeuThrGluTyrPheThr                              145150155160                                                                  PheLysGlnGluCysLysIleLysTyrSerGluArgValTyrGluAla                              165170175                                                                     CysMetGluAlaPheAspSerLeuProLeuAlaAlaLeuLeuAsnGln                              180185190                                                                     GlnPheLeuCysValHisGlyGlyLeuSerProGluIleHisThrLeu                              195200205                                                                     AspAspIleArgArgLeuAspArgPheLysGluProProAlaPheGly                              210215220                                                                     ProMetCysAspLeuLeuTrpSerAspProSerGluAspPheGlyAsn                              225230235240                                                                  GluLysSerGlnGluHisPheSerHisAsnThrValArgGlyCysSer                              245250255                                                                     TyrPheTyrAsnTyrProAlaValCysGluPheLeuGlnAsnAsnAsn                              260265270                                                                     LeuLeuSerIleIleArgAlaHisGluAlaGlnAspAlaGlyTyrArg                              275280285                                                                     MetTyrArgLysSerGlnThrThrGlyPheProSerLeuIleThrIle                              290295300                                                                     PheSerAlaProAsnTyrLeuAspValTyrAsnAsnLysAlaAlaVal                              305310315320                                                                  LeuLysTyrGluAsnAsnValMetAsnIleArgGlnPheAsnCysSer                              325330335                                                                     ProHisProTyrTrpLeuProAsnPheMetAspValPheThrTrpSer                              340345350                                                                     LeuProPheValGlyGluLysValThrGluMetLeuValAsnValLeu                              355360365                                                                     SerIleCysSerAspAspGluLeuMetThrGluGlyGluAspGlnPhe                              370375380                                                                     AspValGlySerAlaAlaAlaArgLysGluIleIleArgAsnLysIle                              385390395400                                                                  ArgAlaIleGlyLysMetAlaArgValPheSerValLeuArgGluGlu                              405410415                                                                     SerGluSerValLeuThrLeuLysGlyLeuThrProThrGlyMetLeu                              420425430                                                                     ProSerGlyValLeuAlaGlyGlyArgGlnThrLeuGlnSerGlyAsn                              435440445                                                                     AspValMetGlnLeuAlaValProGlnMetAspTrpGlyThrThrHis                              450455460                                                                     SerPheAlaAsnAsnThrHisAsnAlaCysArgGluLeuLeuLeuLeu                              465470475480                                                                  PheSerSerCysLeuSerSer                                                         485                                                                           __________________________________________________________________________

What is claimed is:
 1. A method for determining if a cell contains acalcineurin-binding and PKA-binding anchoring protein, the methodcomprising the steps of:lysing the cell to form a lysate; incubating thelysate with a solid support, the solid support having calcineurinmolecules immobilized thereon; washing the solid support; contacting thesolid support with a labeled PKA regulatory subunit that can bind ananchoring protein; washing from the solid support regulatory subunitthat did not bind; detecting regulatory subunit remaining bound on thesolid support; and determining therefrom the presence of acalcineurin-binding and PKA-binding protein in the cell.